Recent studies have indicated that insulin is internalized into target cells and localizes in several intracellular organelles including the nucleus, nuclear membrane, and Golgi. Moreover, these organelles have specific binding sites for insulin. The proposed studies will probe both the nature of these intracellular insulin receptors and whether insulin has direct effects on the cell interior. Recent studies from our laboratory and others have demonstrated that insulin has direct effects on isolated nuclei and nuclear envelopes. In the first series of experiments, using 125I-insulin crosslinking studies and insulin-ferritin, we will probe the nature and location of the insulin receptors found on the nuclear envelope and on the endoplasmic reticulum. In the second series of experiments, we will probe the effects of insulin on nuclear mRNA efflux from isolated nuclei. We will investigate both the efflux of total nuclear mRNA as well as efflux of specific mRNAs for albumin and other proteins using cDNA probes. In addition, direct effects of insulin on nucleoside triphosphatase, the enzyme that regulates mRNA efflux, will be studied and compared both with insulin-like hormones such as the insulin-like growth factors and with epidermal growth factor. It has recently been reported that the interaction of insulin with the plasma membrane insulin receptor produces a small molecular weight protein peptide that may function as a second messenger for insulin. In addition, in studies with nuclear envelopes and isolated nuclei, we will test the effect of this putative second messenger for insulin and compare it to insulin itself. We believe that these types of studies should provide new information concerning the possible direct effects of insulin on nuclear functions.